Attenuation Aβ1-42-induced neurotoxicity in neuronal cell by 660nm and 810nm LED light irradiation

Oligomeric amyloid-β 1–42 (Aβ1–42) has a close correlation with neurodegenerative disorder especially Alzheimer’s disease (AD). It induces oxidative stress and mitochondrial damage in neurons. Therefore, it is used to generate AD-like in vitro model for studying neurotoxicity and neuroprotection against amyloid-β. A low-level light therapy (LLLT) is a non-invasive method that has been used to treat several neurodegenerative disorders. In this study, the red wavelength (660nm) and near infrared wavelength (810nm) at energy densities of 1, 3, and 5 J/cm2 were used to modulate biochemical processes in the neural cells. The exposure of Aβ1–42 resulted in cell death, increased intracellular reactive oxygen species (ROS), and retracted neurite outgrowth. We showed that both of LLLT wavelengths could protect neurons form Aβ1-42-induced neurotoxicity in a biphasic manner. The treatment of LLLT at 3 J/cm2 potentially alleviated cell death and recovered neurite outgrowth. In addition, the treatment of LLLT following Aβ1–42 exposure could attenuate the intracellular ROS generation and Ca2+ influx. Interestingly, both wavelengths could induce minimal level of ROS generation. However, they did not affect cell viability. In addition, LLLT also stimulated Ca2+ influx, but not altered mitochondrial membrane potential. This finding indicated LLLT may protect neurons through the stimulation of secondary signaling messengers such as ROS and Ca2+. The increase of these secondary messengers was in a functional level and did not harmful to the cells. These results suggested the use of LLLT as a tool to modulate the neuronal toxicity following Aβ1–42 accumulation in AD’s brain.


Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disorder in elderly. The etiology of AD is remained unknown but believed to be correlated with the abnormal and MMP production or because of direct stimulation of Ca 2+ transporters [20,21]. However, the therapeutic effect of red and NIR LLLT on Aβ 1-42 -induced toxicity in neuron has not been clearly established. This study therefore focuses on the effects of LLLT on Aβ 1-42 -induced toxicity in neuronal cell and other related photoinduced pathways that will elucidate how to apply LLLT effectively for the treatment of AD and other neurodegeneration.
In this study, we examined the impact of LLLT on Aβ 1-42 -induced toxicity in RA/BDNFdifferentiated SH-SY5Y cells. Our investigation focused on the possible signaling pathways that contribute to the recovery of neurite retraction and viability. We investigated the effect of 660 and 810 nm light-emitting diodes (LED) at the varying intensities of 1, 3, and 5 J/cm 2 . Our findings indicate that LLLT significantly attenuated Aβ 1-42 -induced neurotoxicity and cell death by modulating mitochondrial activity and promoting neurite outgrowth. Remarkably, in contrast to Aβ 1-42 toxicity, both 660 and 810 nm light provoked an elevation of ROS and intracellular Ca 2+ without exerting any toxic effects on the cell nor inducing cell death. It is worth noting that both wavelengths also modulated the mitochondrial depolarization.

SH-SY5Y cell culture
SH-SY5Y neuroblastoma cells were maintained at 37˚c in a humidified atmosphere of 5% CO 2 . Cells were grown in a mixture medium of 1:1 Dulbecco Modified Eagle Medium (DMEM) and Ham's F12 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/ antimycotic. Culture medium was aspirated every 3 days and cells were passaged once they reached 80% confluence using 0.25% Trypsin-EDTA solution.

SH-SY5Y differentiation
SH-SY5Y cells were differentiated following the differentiation protocol developed by Forster, J. I., et al. [10]. Briefly, the neuronal differentiation was carried out in two steps using sequential chemical induction of RA in full medium for 5 days, followed by BDNF in serum-free medium for 2 days. RA/BDNF differentiated SH-SY5Y cells will be referred as neuron-like cells in this work. After cell differentiation, the differentiated neurons were incubated with Aβ 1-42 oligomers for 1 day, and subsequently irradiated with LLLT for 7 days. The media containing Aβ 1-42 were changed every two days. with a 10 min-sonication in a water bath sonicator. The Aβ-HFIP solution was then dried with nitrogen gas to obtain a clear thin peptide film at the bottom of the tubes. Dried peptide films were stored at -20˚C until use. Prior to each experiment, the peptide films were thawed to RT, resuspended in 100% anhydrous Dimethyl Sulfoxide (Sigma-Aldrich, MO, USA) to a final concentration of 5mM, vortexed thoroughly, and sonicated for 10 min. The Aβ 1-42 solution was further diluted in ice-cooled DMEM Phenol red-free medium to acquire a final concentration of 10 μM.

Low-level light irradiation
An AlGaInP light-emitting diodes (LEDs) at 660 nm with 250 mW output power and AlGaAs LED at 810 nm with 340 mW output power. The LEDs were used by focusing the beam from the LEDs planar array on the top of the culture plate. This study uses MATLAB simulation to design LEDs planar array with a sufficient intensity distribution of light. The LEDs array was initially performed by setting the system to be considered and Cartesian coordinates, see S1-S3 Figs. 660 nm LED and 810 nm LED were placed on the irradiating plane with an output of a constant power density at 5 mW/cm 2 . The differentiated neurons were irradiated with a power density of 5 mW/cm 2 for different time periods to achieve energy densities of 1, 3, 5 J/ cm 2 . This experiment was designed to realize the effect of light over an irradiation time of 7 days (pictorially described in Fig 1).

Viability assay
The trypan blue dye exclusion test was used to determine the number of viable cells present in a cell suspension. A viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm. To measure cell viability, the 0.4% w/v of trypan blue dye was added to the cell suspension and thoroughly mix by pipetting. The mixture cells were immediately counted by using a hemocytometer. The percentage of viable cells was calculated by dividing the number of viable cells with the number of total cells and then multiplying with 100.

Measurement of intracellular reactive oxygen species (ROS)
To quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2',7'dichlorodihydrofluorescein diacetate (DCFH-DA) staining modified from the instruction of a previous study [23]. After LLLT, all experimental groups were incubated with 10 μM

Measurement of mitochondrial membrane potential (MMP)
Mitochondrial membrane potential was measured using the cationic JC-1 dye as a sensitive fluorescent probe according to the manufacturer's protocol. In healthy cells with a normal MMP, the JC-1 dye can enter into the mitochondria and accumulate in the energized and negatively charged mitochondria, spontaneously forming red fluorescent J-aggregates. By contrast, in unhealthy or apoptotic cells, the inside of mitochondria is less negative due to increased membrane permeability and consequent loss of electrochemical potential. Thus, JC-1 enters the mitochondria to a lesser extent [24].
After LLLT, cells were incubated with 500 μl JC-1 staining solution at 37˚C for 30 min. Following the incubation, the cells were washed once with the warm phosphate-buffer saline (PBS). Finally, the PBS was added into the cultures and then the fluorescence was observed with a fluorescence microscope (Cytell TM ) using a dual-bandpass filter. Red fluorescence appears under an emission at 590 nm, while green fluorescent appears under an emission at 530 nm.

Measurement of intracellular calcium
Intracellular Ca 2+ was determine using the Fluo-3/AM dye, as an indicator of intracellular Ca 2 + in living cells. After LLLT, the cells were then trypsinized, washed 3 times with PBS, and resuspended in PBS. Subsequently, cell suspensions (100 ml) for Ca 2+ analysis were loaded with 5 mM Fluo-3/AM for 30 min at 37˚C in the dark, washed once with PBS to remove the excessive Fluo-3/AM. PBS, replacing Fluo-3/AM, served as a negative control. Finally, the cells for each example were resuspended in 1 ml PBS, followed by adding the suspension into a 96-well plate (150 ml/well). Fluorescent intensity at 535 nm was recorded using an excitation wavelength at 488 nm with a microplate reader (Infinite1 M200, TECAN).

Software preparation and image analysis
Analysis of neurite outgrowth was performed with ImageJ software according to the protocol outlined by Boulan, Benoit, et al. with some modification [25,26]. First, all images were prepared by optimizing Phase/Contrast and removing the image background. Next, neurite outgrowth was measured by tracing all neurites with NeuronJ toolbar.

Statistical analysis
All graphs represent mean ± standard error values calculated from data obtained from at least three independent experiments, each of which was performed in triplicate. R program was used to perform one-way analysis of variance (ANOVA) and t-test. Serial or multiple comparisons were conducted using analysis of variance and post-hoc testing, or the Kruskal-Wallis test, for parametrically and nonparametrically distributed values, respectively. A Pvalue < 0.05 was considered to reflect statistical significance.

The exposure of oligomeric Aβ 1-42 induced neurotoxicity in differentiated neurons
In this study, neuroblastoma SH-SY5Y cell line was utilized the neurotoxicity against oligomeric Aβ 1-42 . The differentiated neurons were treated with Aβ 1-42 at concentration of 1 and 10μM for 7 days. The morphological study, cell number and neurite outgrowth were observed at day 1, 3, and 7 to determine Aβ 1-42 -induced neurotoxicity. The results showed that Aβ 1-42 treatment decreased cell number and neurite outgrowth in a dose-and time-dependent manner (Fig 2). The cell number decreased to 90.14 ± 17.13, 74.68 ± 10.68, and 66.67 ± 3.29 following 1μM Aβ 1-42 treatment at day 1, 3, and 7, respectively. Whereas it decreased to 39.75 ± 6.36, 28.00 ± 5.24, and 3.25 ± 1.53 following 10μM Aβ 1-42 treatment at day 1, 3, and 7, respectively. The morphological changes were also observed in differentiated cultures in the first day of incubation, especially in 10μM Aβ  . The neurite outgrowth rapidly diminished and could not be determined since day 3 (Figs 2B and 3). We found that 1μM Aβ 1-42 exhibited lower toxicity on neurite outgrowth retraction. The relative neurite outgrowth decreased to 71.67 ± 8.14, 45.90 ± 8.40, and 23.04 ± 4.34% following 1, 3, and 7 days treatments. These results suggested that the neurite retraction was rapidly observed and could be used as a parameter to

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Attenuation Aβ 1-42 -induced neurotoxicity in neuronal cell by 660nm and 810nm LED light irradiation monitor neurotoxicity against Aβ 1-42. Following the results from this experiment, the dose of 1μM Aβ 1-42 treatment for 1 day was, therefore, used in further study.

LLLT attenuated Aβ 1-42 -induced neurotoxicity and recovered neurite outgrowth
To assess the neuroprotective effects of LLLT, differentiated neurons were induced with1μM Aβ 1-42 for 1 day then further treated with LLLT for 7 days (once a day). The LEDs planar array of 660 nm and 810 nm, constant power density of 5 mW/cm 2 , and energy density of 1, 3, and 5 J/cm 2 were used for irradiation. Based on the ability of LLLT to modulate mitochondrial function, we therefore measured cell viability of cell by using trypan blue exclusion assay instead of using MTT assay. The result showed that both of LLLT did not cause noticeable toxic to differentiated neurons, while both LLLT could attenuate Aβ 1-42 -induced neurotoxicity. The neuroprotective effect was observed in a biphasic manner at 3 J/cm 2 with the viability of 72.43% ± 6.10 and 72.42% ± 7.20 at 660 nm and 810 nm, respectively (Fig 4).
In addition to the recovery of cell viability, the percentage of neurite outgrowth and length of neurite were monitored. We found that the retraction of neurite outgrowth upon the presence of Aβ 1-42 was recovered by both LLLT irradiations. The results showed that Aβ 1-42 reduced neurite outgrowth more than 80% comparing with negative control, while both LLLT could significantly recover the neurite outgrowth number and neurite length (Fig 5). The effects of both LLLT on neurite outgrowth and elongation were in a biphasic manner with a peak at 3 J/cm 2 . As shown in Fig 5A and 5B, 660 nm and 810 nm LLLT at 3 J/cm 2 could recover neuron outgrowth from Aβ-induced toxicity from about 20% to 80.44% ± 6.14 and 75.91% ± 3.39, respectively. Besides, 3 J/cm 2 of 660 nm and 810 nm LLLT alone minimally alter neurite length to 104.61% ± 10.05 (P-value = 0.724) and 106.54% ± 7.62 (Pvalue = 0.514), respectively comparing with negative control (Fig 5C and 5D). We found that LLLT attenuated the neurite outgrowth retraction of differentiated neuron under the absent of Aβ 1-42 . This supported that LLLT is a non-invasive therapeutic method that is not harmful to the cells and can be used to protect neurons from Aβ 1-42 toxicity.

LLLT attenuated the mitochondrial depolarization upon Aβ 1-42 treatment
Based on the mitochondrial modulation capacity of LLLT, in this study we measured the MMP upon Aβ 1-42 and LLLT exposure. MMP is the change of potential between two layers. As a result of calcium influx, it results in increasing MMP (depolarization). In general, MMP is monitored by JC-1 dye. It indicates by the shift from green fluorescence to red fluorescence. Thus, the depolarization is indicated by the decrease of red/green fluorescence intensity ratio. Previous study revealed that the oxidative stress led to the cytotoxicity by disrupting MMP and stimulate cell apoptosis. We showed that Aβ 1-42 oligomer could induce cytotoxicity as similar as H 2 O 2 (known potential oxidant). The relative intensity ratio of red/green fluorescence were reduced to about 34.60 ± 5.07% (P<0.01) and 33.68 ± 5.30% (P<0.01) in Aβ 1-42 and H 2 O 2 , respectively (Fig 6). The exposure of LLLT in the absent of Aβ 1-42 did not statistically alter the fluorescence intensity ratio as well as cell viability. Interestingly, the LLLT irradiation following Aβ 1-42 exposure had ability to attenuate the mitochondrial depolarization about 54.26 ± 7.89% in 660 nm and 61.10 ± 2.50% in 810 nm comparing to Aβ 1-42 alone. Previous study showed that LLLT had modulated the mitochondria by interacting with photo-acceptors such as cytochromes, water, lipids, S-nitrosylated nitric oxide (NO) and transient receptor potential channels (TRPC) for Ca 2+ . These interaction occurred through various pathways [27]. Thus, the protective effect of LLLT may be due to the alteration of ions across mitochondrial inner membrane which attenuated the Aβ 1-42 induced-mitochondrial depolarization.

LLLT irradiation diminished the ROS production upon Aβ 1-42 exposure
Previous study revealed that Aβ 1-42 -induced neural toxicity through the elevation of oxidative stress. We found that ROS, determined by DCFH-DA staining, rise to 320.30 ± 15.02%

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following Aβ 1-42 exposure. The elevation of ROS upon Aβ 1-42 was similar to H 2 O 2 , and both conditions rise the intracellular ROS to 3.20 ± 0.15 folds (P<0.01) and 4.12 ± 0.68 folds (P<0.01), respectively comparing with negative control (Fig 7B). We showed that the irradiation of LLLT both wavelengths could attenuate the elevation of intracellular ROS upon Aβ 1-42 treatment. The LLLT of 660 nm and 810 nm showed therapeutic effects by significantly reduced ROS levels to 0.91 and 1.23 folds, respectively. Interestingly, both LLLT itself could also stimulate intracellular ROS. The LLLT of 660 nm increased ROS production to 1.47 folds, while 810 nm increased ROS to 1.84 folds (P-value = 0.010). The increase of ROS in LLLT alone did not cause cell death. Thus, we believed that ROS may be one of the players that act as a secondary messenger to modulate the cellular response. This level of ROS was lower than ROS from Aβ 1-42 and H 2 O 2 treatments. While the LLLT treatment following Aβ 1-42 did not exert the ROS level, in contrast, in lowered the intracellular ROS and recovered cell death and neurite outgrowth (Figs 4 and 5).

LLLT lessen the intracellular calcium and protected neuron from Aβ 1-42induced toxicity
In this study intracellular calcium (Ca 2+ i ) was monitored by Fluo-3 fluorescence intensity. Due to the transient response of Ca 2+ i upon LLLT irradiation, we monitored the Ca 2+ i at 10 minutes after exposure to the light. We showed that LLLT alone could induced Ca 2+ i to 0.389 ± 0.003 and 0.458 ± 0.178 of LLLT 660 and 810 nm, respectively. While the presence of

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Aβ 1-42 for 1 day raised Ca 2+ i to about 9 folds. We found that the exposure of LLLT once a day for four days could retain Ca 2+ i to about 6 folds in both LLLT wavelengths, which were significantly lower than Aβ 1-42 exposure alone (Fig 8). We found that LLLT-induced Ca 2+ i elevation effect was transient. The concentration of Ca 2+ i was diminished to non-lethal level within one hour, whereas induction of Ca 2+ i following Aβ 1-42 exposure were prolonged. We believed that calcium ion may be one of the players that stimulated cellular protection process. LLLT alone did not harmful to the cell, while the exposure of LLLT following the cellular damage from Aβ 1-42 exposure could attenuate neurotoxicity.

Discussion
Amyloid plaques have been used as one of the histopathological hallmarks of AD (post-mortem) for many decades. In addition, PET amyloid-β imaging has also been used to diagnose AD patients together with memory assessment while they are still alive. Amyloid-beta (Aβ), especially the soluble oligomeric forms of the Aβ 1-42 peptide, plays critical roles in AD pathogenesis. It exhibits greater toxicity in neuronal cells than their monomer or fibril forms [28,29]. The accumulation of Aβ 1-42 oligomer in the brain induced the oxidative stress, mitochondrial dysfunction, and chronic inflammation. This leads to neural network dysfunction and neurodegenerative diseases [30][31][32][33]. Thus, oligomeric Aβ 1-42 had been used to generate AD in vitro model. In this study, we induced neuroblastoma cell with RA and BDNF toward differentiated neuron stage [10]. The differentiation model showed neuron-like cell morphology, which suited for studying neurite outgrowth and neural toxicity for Aβ 1-42 -induced AD model. Following the differentiation, neurons were exposed with oligomeric Aβ 1-42 for 24

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hours prior to LLLT irradiation. Cells were treated with LLLT once a day for 7 days in the presence of Aβ 1-42 . We found that LLLT exhibited the therapeutic effects in Aβ 1-42 -induced neural toxicity.
Neurite outgrowth was used as an early marker to investigate the morphological changes and cell death. This is an important process in neuronal development, particularly the in vitro neural networks formation and in vivo nerves regeneration. We found that Aβ 1-42 had neurotoxicity in a concentration and time-dependent manner. Following the treatment of oligomeric Aβ 1-42 , intracellular ROS and Ca 2+ across while mitochondrial were depolarized. Along with the molecular responses, we observed the axon destruction and cell death in our in vitro model. In addition, these observations also observed in AD patients and animal models in several previous studies [34][35][36][37][38].
The molecular mechanisms underlying neuroprotective effects of LLLT might associated with the modulation of intracellular ROS and Ca 2+ . Previous study revealed that, Aβ could accelerate the mitochondria swelling by Ca 2+ overload through the opening of the mitochondrial permeability transition pore (mPTP) opening [39]. The opened mPTP were induced by

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Ca 2+ signaling and ROS which subsequently led to mitochondrial depolarization [40][41][42][43]. Previous study in the hippocampal neuron and astrocytes revealed that the arise of oxidative stress and mitochondrial dysfunction related to the activation of NADH oxidase [44]. In this study, oligomeric Aβ 1-42 could induce intracellular ROS about 3 folds and changed mitochondrial health (determined by JC-1 staining) about 35%. This ROS overproduction and collapse of mitochondrial membrane potential were similarly to the treatment of H 2 O 2 , a membrane-permeable reactive oxygen species.
Mitochondria is the critical energy resource of the living cells. It is known to be the primary photoreceptor of LLLT. Several previous studies showed that red and NIR LLLT activated ATP production, reduced oxidative stress, and enhanced the sprouting of neurite outgrowth [45][46][47]. The photo-biomodulation may be described by the transfer of electron from LLLT that modulate redox cycling and secondary messenger accumulation. LLLT has been shown to enhance mitochondrial respiration and activate the redox-sensitive NF-kB signaling via the generation of ROS [48]. In addition, NIR light treatment effectively reduced synaptic vulnerability to Aβ oligomers by moderating the binding of Aβ on synaptosomes and increasing synaptic mitochondria in vivo [49].
LEDs has been reported as a good light source for emitting red and NIR wavelengths. Due to the interference phenomenon and the excellent tissue scattering characteristics of light at these wavebands, LED had high efficacy of cell penetration [50]. However, the different wavebands could display different bio-stimulation mechanisms. LLLT at 980 nm targeted temperature-gated calcium ion channels, while 810 nm targeted mitochondrial cytochrome c oxidase inhuman adipose-derived stem cells [51]. Neurite retraction in N2a cell following blue-light (473 nm) exposure could be recovered by spotting the red-light (650 nm) on the soma near the junction of retraction site. Furthermore, green-light (550 nm) also had protective effect but lower than red-light. Previous study showed that the stimulation of neurite extension by redlight was associated with the Ca 2+ influx, actin propagation and myosin II inhibition [52]. In addition, NIR light (808 nm) could promote the neuronal growth of trigeminal ganglion neurons through the activation of mitochondria [53,54]. Moreover, LLLT with 660 nm and 810 nm at the dose of 3 J/cm 2 had been reported to increase ATP, raised MMP, and stimulated proliferation rate of hASCs in a relatively similar fashion [55]. Although red and NIR light have been reported to activate cell function, the effects of these wavebands on the cellular level of neurons are still unclear, and the optimal conditions for fluences and irradiance of light exposure were varied depending on the cell type and pathological condition. Our findings showed that a density of 3 J/cm 2 could recover viability, neurite outgrowth and neurite elongation upon the presence of Aβ 1-42 . However, the higher density of 5 J/cm 2 and the lower density of 1 J/cm 2 showed lower neuroprotective effects. These effects were appeared as biphasic manner, which were previously observed in other study [56]. LLLT at 810 nm with fluences of 3 J/ cm 2 showed a biphasic-dose response, which can induce a significant increase in intracellular calcium, ATP, and MMP, as well as stimulate modest levels of ROS that activate signaling pathways. However, these effects decline at higher fluences of 30 J/cm 2 , and can result in a significant increase in ROS levels that induce impaired mitochondrial function and initiate neural death. Previous research has also shown that LLLT at 810 nm with 3 J/cm 2 can elevate MMP and ROS in normal neurons. Conversely, in oxidatively-stressed cells, LLLT can increase MMP and reduce ROS, thereby protecting against neural death [57]. In this study, both wavelengths; 660 nm and 810 nm were not only restored of neurite outgrowth but also attenuated the Aβ-induced toxicity at all light doses. They exhibited unnoticeable toxicity to the differentiated neurons (Figs 4 and 5). Based on the findings, we suggested that if the irradiation time was too long, the heat was accumulate and led to cellular damage. On the other hand, if the irradiation time was too short, the treatment would be ineffective due to an insufficient photon density to reach the target chromophore. Thus, the therapeutic use of LLLTs should be restricted their energy density and irradiation time.
We found that both 810 nm and 660 nm LLLT significantly relieved Aβ 1-42 -induced neurotoxicity. They improve viability, neurite outgrowth, and neurite elongation upon the irradiation for 3 days, although the LLLT at 810 nm may have greater potential for neuroprotective effects compared to 660 nm (Fig 9). We believed that both red and NIR wavebands could similarly induce mitochondrial photon absorption, intracellular signal transduction and the downstream cellular responses. Based on our observation red and NIR LLLTs was able to recover MMP and improve in cell viability. We showed that the increase of intracellular ROS and Ca 2+ upon Aβ 1-42 exposure induced the opening of the mPTP and subsequently led to mitochondrial dysfunction. The high and prolonged increase in cytoplasmic Ca 2+ levels, up to 500 nM or five-fold over 20 to 30 minutes, was required for neuron death via excitotoxicity [58], while a transient elevation of Ca 2+ levels from LLLT irradiation may responsible for the bio-stimulatory effects [45]. Previous studies showed that the prolonged opening of the mPTP were associated with a progressive rise of intracellular ROS levels, and elevated Ca 2+ level. These resulted in the degradation of the mitochondria and ultimately program cell death [59][60][61][62].
Based on our findings, we suggested that LLLT at 810 nm and 660 nm could stimulate the transient induction of ROS and Ca 2+ , which were present under the sub-lethal amount or lower than the level that causes neuron excitotoxicity. The increase of intracellular ROS and Ca 2+ levels upon exposure to Aβ 1-42 was associated with a reduction in MMP, rendering neurons vulnerable to toxicity, physiological changes, and cell death. Conversely, LLLT has the potential to produce the signaling of ROS and Ca 2+ , promoting cellular function and

Conclusions
The effects of low intensity 660 nm and 810 nm light on Aβ 1-42 toxicity in the differentiated neurons have been demonstrated in this study. In this study, LLLT at 660 nm and 810 nm showed a biphasic dose response at 3 J/cm 2 that could protect Aβ 1-42 toxicity by recovering neurite outgrowth and cell viability. LLLT could generate the signaling of ROS and Ca 2+ which in turn promoted downstream cell signaling pathway and provide the beneficial effects for mitochondrial activity (Fig 10). However, the molecular responses upon LLLT have not been determined in this study and should be investigated further in the future study.

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LED arrays when an average energy density at 3 J/cm 2 is required. (TIFF)